NZ722156A

NZ722156A - Ex vivo antibody production

Info

Publication number: NZ722156A
Application number: NZ722156A
Authority: NZ
Inventors: Helden Pauline Maria Wilhelmina Van; Mark Jeroen Kwakkenbos; Hergen Spits; Tim Beaumont
Original assignee: Kling Biotherapeutics B V
Priority date: 2013-12-24
Filing date: 2014-12-24
Publication date: 2021-08-27
Also published as: EP3087177B1; JP2017500052A; IL246409B; BR112016014881A8; ES2927905T3; AU2014370516B2; CN106029873A; HUE060145T2; CA2934660C; DK3087177T3; US20200149007A1; BR112016014881A2; EP4141108A1; JP6832705B2; EP3087177A1; AU2014370516B9; CA2934660A1; IL246409A0; US12084681B2; AU2014370516A1

Abstract

The present invention provides means and methods for producing improved ex vivo rabbit B cell cultures with a short doubling time of less than 20 hours or less. The methods comprise inducing, enhancing or maintaining expression of Bcl-6 and at least one anti-apoptotic nucleic acid comprising a gene of the Bcl-2 family in a rabbit B cell. The invention further relates to the use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein for introducing a nucleic acid molecule encoding Bcl-6 and the anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell. The invention further relates to rabbit B cells and ex vivo rabbit B cell cultures obtained by such methods.

Description

Title: Ex vivo antibody production The invention relates to the fields of medicine, molecular biology and immunology.

Ex vivo B cell cultures are important tools for producing antibodies, preferably monoclonal antibodies. Monoclonal antibodies (mAbs) represent multiple identical copies of a single antibody molecule, which copies bind to antigens with the same affinity and promote the same or functions. Amongst the benefits of mAbs is their specificity for the same e on an antigen. This specificity confers certain clinical advantages on mAbs over more conventional treatments while offering ts an effective, well-tolerated therapy option with generally low side effects. Moreover mAbs are useful for biological and medical research.

A conventional approach for obtaining mAbs is hybridoma technology, n a B cell is fused with a myeloma cell in order to form hybrid antibody producing cell lines (hybridomas). However, hybridoma technology with human B cells has not been very successful because the resulting hybridomas are unstable. ile, an improved technology has been developed wherein ex vivo B cell cultures are produced with a prolonged replicative life span ().

This technology involves human ex vivo cultures wherein Bcl-6, together with 1 and/or an anti-apoptotic nucleic acid, are expressed in the B cells. This improves the replicative life span of these B cells. lly, human B cells are cultured in order to obtain human mAbs. Human mAbs are preferred for eutic applications in humans due to the lower immunogenicity as compared to antibodies of other species. Using the technology of , ex vivo human B cell cultures with a mean doubling time of about 25-36 hours are obtained.

In order to commercially produce mAbs of interest, such as therapeutic mAbs, it is advantageous to use B cell cultures n the B cells have a short ng time. A short doubling time is also very important in therapeutic approaches like cancer therapy, for instance when a non-human mammal is immunized with cancer cells of a patient, where after cancer-specific B cells are harvested from the animal and used for ex vivo antibody production. Since such antibodies are a tailor-made medicine for the individual patient, they should be ed as fast as possible so that the t can start his/her Ab therapy as soon as possible. Such antibodies that are specific for an individual’s tumor cannot be produced beforehand.

It is one of the objects of the present invention to provide means and methods for producing improved ex vivo B cell cultures with a shorter doubling time; and or to at least provide the public with a useful choice.

The present invention provides the insight that B cell cultures with a shorter doubling time, as compared to the B cell cultures disclosed in WO 67046, are obtained when rabbit B cells are used. Whereas commonly used B cells such as human B cells, murine B cells and llama B cells lly have a doubling time of 25-36 hours, the present inventors have surprisingly found that rabbit B cell cultures can be obtained with a ng time of 20 hours or less. This insight allows significant faster production of antibodies of interest, resulting in a higher yield within a given time frame, which is ularly valuable for cial antibody production and therapeutic applications.

Summary of the ion In a first aspect the present invention provides a method for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less, the method comprising: - inducing, enhancing or ining expression of Bcl-6 in a B cell, - inducing, enhancing or maintaining expression of at least one antiapoptotic nucleic acid molecule comprising a gene of the Bcl-2 family in said B cell, characterized in that said B cell is a rabbit B cell.

In a second aspect the present invention provides a method for increasing the replicative life span of a rabbit B cell, the method sing: - inducing, enhancing or maintaining expression of Bcl-6 in a rabbit B cell, and - inducing, enhancing or ining expression of at least one antiapoptotic nucleic acid comprising a gene of the Bcl-2 family in said B cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, with at least one anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family, or with a combination thereof, via transduction with a gene ry vehicle that comprises the extracellular domain of a gibbon ape leukemia virus (GALV) pe protein or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein.

In a third aspect the present invention provides a use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV pe protein, for ucing a nucleic acid molecule encoding Bcl-6 and at least one anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell.

In a fourth aspect the present invention provides a method for obtaining antibodies, comprising: - ng, enhancing or maintaining expression of Bcl-6 in a rabbit B cell; - inducing, enhancing or maintaining expression of at least one antiapoptotic nucleic acid molecule comprising a gene of the Bcl2- family in said B cell; - culturing said B cell ex vivo; and - harvesting dies produced by said B cell within 7-14 days.

In a fifth aspect the present invention provides a rabbit B cell, which is bound via the extracellular domain of a GALV envelope protein, or via a protein that has at least 70% sequence identity with the ellular domain of a GALV envelope protein, to a gene delivery vehicle that comprises a nucleic acid sequence encoding Bcl-6 and an anti-apoptotic nucleic acid sequence sing a gene of the Bcl-2 family.

In a sixth aspect the present invention provides an isolated or recombinant rabbit B cell comprising: - a non-rabbit anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 , and - a bbit nucleic acid molecule encoding Bcl-6.

In a seventh aspect the present invention provides an ex vivo rabbit B cell culture comprising the rabbit B cells according to the fifth or sixth aspects, which has a mean ng time of 20 hours or less.

In an eighth aspect the present invention provides an ex vivo rabbit B cell culture when obtained by a method according to the first .

In a ninth aspect the present invention provides a use of a gene delivery vehicle comprising the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, and a c acid ce encoding Bcl-6 and at least one antiapoptotic nucleic acid sequence comprising a gene of the Bcl-2 family, for increasing the replicative life span of a rabbit B cell.

Also described is a use of a rabbit B cell for obtaining an ex vivo B cell e with a mean doubling time of 20 hours or less. Ex vivo rabbit B cell cultures described herein are typically obtained by expression of Bcl-6, or a rabbit homologue thereof, and an anti-apoptotic nucleic acid molecule in a rabbit B-cell.

Further described is therefore a method for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less, the method comprising: - inducing, ing and/or maintaining expression of Bcl-6, or a rabbit homologue thereof, in a B-cell and - ng, enhancing and/or maintaining expression of an poptotic nucleic acid molecule in said B-cell, characterized in that said B cell is a rabbit B cell.

Preferably, ex vivo rabbit B cell cultures are produced with a mean doubling time of less than 20 hours. More preferably, said mean doubling time is less than 19 hours or even less than 18 hours. A shorter doubling time allows faster and higher dy production, which enhances the time to - and efficacy of - testing and screening for a desired antibody and isolation and/or identification of antibodies of interest. Moreover, if mAbs need to be developed for an individual patient, the shorter doubling time of rabbit B cells allows for a quicker start of the patient’s specific mAb y.

A method described herein, using rabbit B cells, thus provides the advantage that antibody can be obtained, tested, identified, isolated and/or produced ex vivo within a r time frame as compared to currently known human, murine or llama B cell cultures.

The fact that the present disclosure describes a B cell culture with a short doubling time provides the advantage that a sufficient quantity of antibody can be obtained within a shorter period of time as ed to existing methods. For instance, in a method as disclosed in a tion of B cells obtained from a human individual is stabilized using Bcl-6 and an anti-apoptotic nucleic acid (or compounds increasing the expression of such nucleic acids) and subsequently cultured. This results in stabilized human B cells, which are capable of both erating and producing antibody. During culturing, the stabilized B cells produce antibody, which is secreted into the culture medium. Subsequently, these dies are preferably tested for a desired specificity (and/or affinity). For t test procedures, an antibody concentration of at least 100 ng/ml culture medium is lly required. After 15-20 days of culturing stabilized human B cells, such minimal antibody concentration is obtained. Therefore, using human B cell cultures, antibody is harvested at least 15-20 days after ng the culture, typically around day 20. Llama B cells have a similar growth rate as human B cells, so that if a llama B cell culture is used, antibody is also typically harvested at least 15-20 days after starting the culture. With murine B cells, which have a longer doubling time, dies with a minimal concentration of 100 ng/ml are typically obtained after more than 20 days.

After testing the antibodies, the corresponding B cells of interest are often ed and isolated for further use. Given the fact that antibody testing normally takes about three days, human or llama B cells of interested are lly selected and isolated after 18-23 days from the start of the B cell culture, whereas murine B cells of interest are typically ed and isolated after more than 23 days. The isolated B cells are then further cultured. A B cell culture with human, llama or murine B cells of interest is thus typically obtained after about three weeks from the start of the B cell culture. With a method described herein, however, an antibody concentration of at least 100 ng/ml is already ed after 11-12 days.

Hence, antibody can now already be harvested 11-12 days after starting the B cell culture, whereas one had to maintain a human (or llama) B cell culture for at least -20 days before harvesting antibody. If the testing procedure takes three days, rabbit B cells of interest are thus selected and isolated within 14-15 days from the start of the B cell e, which is significantly faster as compared to the situation wherein human or murine B cells are cultured. In conclusion, whereas it typically takes about three weeks for obtaining a human, llama or murine B cell culture which produces a sufficient concentration of antibody, with the insight of the present disclosure a B cell culture with rabbit B cells producing a ient Ab concentration is already obtained after two weeks. This is a major advantage over ng methods. One embodiment described herein relates to a method for obtaining antibodies, ably for use in one or more testing assays requiring a minimal antibody concentration of at least 100 ng/ml, the method comprising: - inducing, enhancing and/or maintaining expression of Bcl-6 in a rabbit B-cell; - inducing, enhancing and/or maintaining expression of an anti-apoptotic nucleic acid molecule in said B-cell; - culturing said B cell ex vivo; and - harvesting antibodies produced by said B cell within 10-14 days, preferably within 11-12 days. Said harvested antibodies are preferably tested using one or more assays requiring a minimal antibody concentration of at least 100 ng/ml.

As described above, the obtained antibodies are typically used for testing for a desired specificity and/or ty. Current test methods often require a minimal antibody tration of 100 ng/ml, but if more sensitive detection methods are used, the antibodies can be harvested earlier. er the sensitivity of the test method, using rabbit B cells with a method according to the present disclosure, the required l antibody concentration is obtained earlier as compared to the use of currently known human, llama or murine B cells, due to the significant faster doubling time of rabbit B cells. For instance, if a minimal antibody tration of only 30 ng/ml is required, instead of 100 ng/ml, this concentration is typically reached using human B cells after 13-18 days from the start of the B cell culture, whereas a rabbit B cell e would only need 9-10 days to obtain this minimal antibody concentration. Thus, again, antibody testing and isolation of B cells of interest can be performed earlier. In practice, the current ors obtain and test the rabbit antibodies within 7-14 days from the start of a B cell culture. Before the present disclosure, ex vivo B cell cultures allowing antibody testing at significant earlier stages as compared to ex vivo human B cell cultures were not available.

Further bed is therefore a method for obtaining antibodies, the method comprising: - ng, enhancing and/or maintaining expression of Bcl-6, or a rabbit gue thereof, in a rabbit B-cell; - ng, enhancing and/or maintaining expression of an anti-apoptotic c acid molecule in said rabbit B-cell; - culturing said B cell ex vivo; and - harvesting antibodies produced by said B cell within 7-14 days, preferably within 9-12 or 9-10 days. Said harvested antibodies are preferably tested using one or more assays requiring a minimal antibody concentration of about 30 ng/ml.

As used herein, the term "rabbit B cell" means a B cell that has been obtained from a rabbit, or a B cell that originates from a rabbit B cell. An example of B cells originating from a rabbit B cell is the progeny of a rabbit B cell that is formed after one or more cell on cycles. Such y for instance includes an ex vivo culture of rabbit B cells.

An ex vivo rabbit B cell culture is a culture that contains rabbit B cells and/or progeny thereof. Other kinds of cells may also be present in the culture. For instance, B cell stimulator cells such as CD40 positive L cells and/or EL4B5 cells are typically also present in a B cell culture described herein. Additionally, other kinds of cells, which were also present in a B cell-containing sample, could still be present in a B cell culture. When present in B cell culturing conditions, such non- B cells are typically less capable of proliferating as compared to B cells, so that the number of such contaminating cells will typically decline in time. Preferably, at least 70% of the cells of a rabbit B cell culture are rabbit B cells. More ably, at least 75%, 80%, 85%, 90% or 95% of the cells of said rabbit B cell culture are rabbit B cells. In a particularly preferred ment, rabbit B cells and B cell stimulator cells such as CD40 positive L cells and/or EL4B5 cells are ially the only kinds of cell present in a rabbit B cell culture. ably, the B cells of a rabbit B cell culture described herein are progeny of one original rabbit B cell, so that monoclonal antibodies are produced by the B cell culture.

The term "mean doubling time" is defined herein as the mean time required, starting from a e with a certain original amount of B cells, to obtain a culture with a number of B cells that is two times said original B cell number. Since not every B cell will proliferate at exactly the same rate, mean values are typically used for a B cell culture as a whole.

Bcl-6 s a transcriptional repressor which is required for normal B cell and T cell development and maturation and which is required for the formation of germinal centers. Bcl-6 is highly sed in germinal center B cells whereas it is hardly expressed in plasma cells. Bcl-6 ts differentiation of activated B cells into plasma cells. In a method described herein, Bcl-6 expression product, or the expression product of a rabbit gue thereof, remains present in the rabbit B cells of an ex vivo culture. The ce of Bcl-6, or a rabbit homologue thereof, together with the presence of an anti-apoptotic nucleic acid, prolongs the replicative life span of the B cells. sion of Bcl-6, or a rabbit homologue thereof, is preferably induced, enhanced or maintained by administering a Bcl-6 expression-promoting compound, or a compound that promotes expression of a rabbit homologue of Bcl-6, to the rabbit B cell(s) used for culturing, or by culturing rabbit B cells in the presence of such compound.

Further described is therefore a method according to the present disclosure, comprising: - providing said rabbit B cell with a compound capable of directly or indirectly enhancing expression of Bcl-6, or expression, of a rabbit homologue of Bcl-6; and/or - culturing said rabbit B cell in the presence of a compound capable of directly or indirectly ing expression of Bcl-6, or expression of a rabbit homologue of Bcl- Various compounds capable of directly or indirectly enhancing expression of Bcl-6, or expression of a rabbit homologue of Bcl-6, are known in the art. Such nd for instance comprises a Signal ucer of Activation and Transcription 5 ) protein, or a rabbit homologue thereof, or a functional part or a functional tive thereof, and/or a nucleic acid sequence coding therefore. STAT5 is a signal transducer capable of enhancing Bcl-6 expression.

There are two known forms of STAT5, STAT5a and STAT5b, which are encoded by two different, tandemly linked genes. Administration and/or activation of STAT5, or a rabbit homologue thereof, results in enhanced levels of Bcl-6, or enhanced levels of a rabbit homologue of Bcl-6. Hence, STAT5, or a rabbit homologue thereof, or a functional part or a onal derivative thereof is capable of directly increasing expression of Bcl-6, or expression of a rabbit homologue of Bcl-6.

Described is therefore a method according to the present disclosure comprising providing said rabbit B cell with STAT5, or with a rabbit homologue thereof, or with a functional part or a functional derivative thereof, or ing said rabbit B cell with a nucleic acid molecule ng STAT5, or a rabbit homologue thereof, or a functional part or a functional derivative f, or ing said rabbit B cell in the presence of STAT5, or in the presence of a rabbit homologue thereof, or a functional part or a functional derivative thereof.

The presence of STAT5, or a rabbit homologue thereof, directly increases the amount of Bcl-6, or the amount of a rabbit homologue of Bcl-6. It is also possible to indirectly increase expression of Bcl-6, or expression of a rabbit homologue f.

This is for instance done by regulating the amount of a certain compound, which in turn is capable of directly or indirectly activating STAT5, or a rabbit homologue thereof, and/or increasing expression of STAT5, or expression of a rabbit gue thereof. Hence, in one embodiment the expression and/or activity of endogenous and/or exogenous STAT5, or the sion of a rabbit homologue thereof, is increased. It is for instance possible to indirectly enhance expression of Bcl-6, or expression of a rabbit homologue thereof, by culturing a rabbit B cell in the presence of interleukin (IL) 2 and/or IL4 which are capable of activating STAT5, or activating a rabbit homologue of STAT5, which in turn increases expression of Bcl-6, or expression of a rabbit gue of Bcl-6.

As used herein, the term t homologue" of, for instance, Bcl-6 or STAT5 means a rabbit protein corresponding to Bcl-6 or STAT5, which means that it has a corresponding, r function in rabbit B cells as compared to the function of Bcl- 6 or STAT5 in human B cells.

It is, however, preferred to provide a rabbit B cell with a c acid molecule encoding Bcl-6, or encoding a rabbit homologue thereof, or a functional part or a functional derivative thereof. This way, it is le to directly regulate the concentration of Bcl-6, or the tration of a rabbit homologue thereof, in said rabbit B cell. Also described is therefore a method according to the present sure comprising providing said rabbit B cell with a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homologue of Bcl-6, or a functional part or a functional derivative thereof. In one embodiment, said nucleic acid molecule is constitutively active, meaning that Bcl-6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, is continuously expressed, independent of the ce of a regulator. In another embodiment, said nucleic acid molecule is inducible, meaning that the expression thereof is regulated by at least one inducer and/or repressor. This way, expression of said nucleic acid molecule is regulated at will. For instance, Tet-On and Tet-Off expression systems (for example ® and Tet-Off® Advanced Inducible Gene Expression Systems, Clontech) can be used for inducible sion of a nucleic acid sequence of interest. In these systems expression of the transcriptional activator (tTA) is regulated by the presence (Tet-On) or e (Tet-Off) of tetracycline (TC) or a derivative like doxycycline (dox). In principle, tTA is composed of the Escherichia coli Tet repressor protein (TetR) and the Herpes simplex virus transactivating domain VP16. tTA regulates transcription of a nucleic acid sequence of interest under the control of a tetracycline-responsive t (TRE) comprising the Tet operator (TetO) DNA sequence and a promoter sequence, for instance the human cytomegalovirus (hCMV) promoter. A nucleic acid sequence encoding, for instance, Bcl6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, can be placed downstream of this er.

In the Tet-off system, tTA binds to TRE in the absence of TC or dox and transcription of a c acid ce of interest is activated, whereas in the presence of TC or dox tTA cannot bind TRE and expression of a nucleic acid sequence of interest is inhibited. In contrast, the Tet-on system uses a reverse tTA (rtTA) that can only bind the TRE in the presence of dox. Transcription of a nucleic acid sequence of interest is inhibited in the absence of dox and activated in the presence of dox.

In another ment, ble expression is executed using a hormone inducible gene expression system such as for instance an ecdysone inducible gene expression system (for example RheoSwitch®, New England Biolabs) (Christopherson, K.S. et al. PNAS 89, 6314-8 ). Ecdysone is an insect steroid hormone from for example Drosophila melanogaster. In cells transfected with the ne receptor, a dimer consisting of the ecdysone receptor (Ecr) and retinoid X receptor (RXR) is formed in the presence of an ecdyson agonist selected from ecdysone, one of its analogues such as erone A and ponasterone A, and a non-steroid ecdysone agonist. In the presence of an agonist, Ecr and RXR interact and bind to an ecdysone se t that is present on an expression cassette. Exaperssion of a nucleic acid sequence of interest that is placed in an expression cassette downstream of the ne response element is thus induced by exposing a rabbit B-cell to an ecdyson agonist.

In yet r embodiment of the present disclosure inducible expression is executed using an arabinose-inducible gene expression system (for example pBAD/gIII kit, Invitrogen) (Guzman, L. M. et al. Bacteriol 177, 4121–4130 (1995)).

Arabinose is a monosaccharide containing five carbon atoms. In cells ected with the arabinose-inducible promoter PBAD expression of a nucleic acid sequence of interest placed downstream of PBAD can then be induced in the ce of arabinose.

It is also possible to use (a nucleic acid molecule encoding) a Bcl-6 protein, or a rabbit homologue f, or a functional part or functional derivative thereof, wherein the activity of said Bcl-6 or rabbit homologue or functional part or functional derivative is regulated by at least one inducer and/or repressor. A nonlimiting example is a fusion protein wherein a regulatory element is fused to a sequence encoding at least part of Bcl-6 or a rabbit homologue thereof. For instance, an estrogen receptor (ER) is fused to Bcl-6, resulting in fusion protein ERBcl-6.

This fusion protein is inactive e it forms a complex with heat shock proteins in the cytosol. Upon administration of the exogenous inducer 4 hydroxytamoxifen (4HT), the fusion protein ER-Bcl-6 dissociates from the heat shock proteins, so that the Bcl-6 part of the fusion protein becomes .

As used herein, the term apoptotic nucleic acid molecule" refers to a nucleic acid molecule, which is capable of delaying and/or preventing apoptosis in a rabbit B cell. Preferably, said anti-apoptotic nucleic acid molecule is e of delaying and/or preventing apoptosis in a plasmablast-like rabbit B cell, which is capable of both proliferating and producing dy. Preferably, an anti-apoptotic nucleic acid molecule is used which comprises an exogenous nucleic acid molecule.

This means that either a nucleic acid sequence is used which is not naturally expressed in rabbit B cells, or that an onal copy of a naturally occurring nucleic acid sequence is used, so that expression in the resulting rabbit B cells is enhanced as ed to l rabbit B cells. s anti-apoptotic nucleic acid molecules are known in the art, so that various embodiments are available.

Preferably, an anti-apoptotic nucleic acid molecule is used which is an anti- tic member of the Bcl-2 family because anti-apoptotic Bcl-2 proteins are good apoptosis inhibiters in B cells. Many processes that are controlled by the Bcl-2 family (which family includes both pro- and poptotic proteins) relate to the mitochondrial pathway of apoptosis. The use of poptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, Bclrelated protein A1 (also named Bcl2-A1 or A1), Bcl-2 like 10 (Bcl2L10) and Mcl-1, or a rabbit homologue thereof, or a functional part or functional derivative thereof, is preferred because Bcl-2, Bcl-xL, Bcl-w, A1, Bcl2L10 and Mcl-1 are generally integrated with the outer mitochondrial membrane. They directly bind and inhibit the pro-apoptotic proteins that belong to the Bcl-2 family to t mitochondrial membrane integrity.

A preferred embodiment therefore describes a method according to the present sure, wherein said poptotic nucleic acid molecule comprises an anti-apoptotic gene of the Bcl2 family, preferably Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a rabbit homologue thereof, or a functional part or a functional derivative thereof.

In one embodiment, expression of Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a rabbit homologue thereof, is induced, enhanced or maintained by administering at least one compound, capable of promoting expression of any of these poptotic genes, to rabbit B cell(s), or by culturing rabbit B cells in the presence of such compound(s). Further described is therefore a method according to the present disclosure, comprising: - providing said rabbit B cell with a compound capable of ly or indirectly enhancing sion of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10, or a rabbit homologue thereof; and/or - culturing said rabbit B cell in the presence of a compound capable of directly or indirectly enhancing expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10, or a rabbit homologue thereof.

Preferably, however, a rabbit B cell is described herein with at least one nucleic acid molecule encoding an anti-apoptotic gene of the Bcl2 family, preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, and rabbit homologues thereof, and functional parts and functional derivatives thereof.

This way, it is possible to directly enhance the amount of expression product in said rabbit B cell. Also described is therefore a method ing to the present disclosure, comprising providing said rabbit B cell with at least one nucleic acid molecule encoding an anti-apoptotic gene of the Bcl2 , preferably ed from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, and rabbit homologues thereof, and functional parts and functional derivatives thereof. In one embodiment, said nucleic acid molecule is constitutively active, meaning that said nucleic acid molecule is continuously expressed. In another ment, said nucleic acid molecule is inducible, meaning that the expression thereof is regulated by at least one inducer and/or repressor. Non-limiting examples of inducible c acid expression systems known in the art are described herein before.

In a particularly preferred embodiment said anti-apoptotic nucleic acid molecule encodes Bcl-xL or Mcl-1, or a rabbit homologue f, or a functional part or a functional derivative thereof. ing to the present disclosure, a combination of Bcl-6 and Bcl-xL is ularly well capable of increasing the ative life span of rabbit B-cells, thereby forming long term cultures of the resulting plasmablast-like B-cells. The same holds true for a combination of Bcl-6 and Mcl-1. Most ably, said poptotic nucleic acid encodes Bcl-xL or a functional part or a functional derivative thereof.

A functional part of Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or of a rabbit homologue thereof, is a proteinaceous molecule that has the same capability - in kind, not necessarily in amount - of increasing the replicative life span of a rabbit B cell as compared to natural Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or a rabbit homologue thereof, respectively. Such functional part is for instance devoid of amino acids that are not, or only very little, involved in said capability.

For ce, functional parts of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, or of a rabbit homologue thereof, are defined herein as fragments of BclxL , Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, respectively, or of a rabbit homologue thereof, which have retained the same kind of anti-apoptotic characteristics as full length Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, respectively, or a rabbit homologue thereof (in kind, but not necessarily in amount). onal parts of BclxL , Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or of a rabbit homologue thereof, are typically shorter fragments of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, respectively, or of a rabbit homologue thereof, which are capable of delaying and/or preventing apoptosis in a rabbit B-cell. Such functional parts are for ce devoid of sequences which do not icantly contribute to the anti-apoptotic ty of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10. A functional part of Bcl-6, or of a rabbit gue thereof, is typically a shorter fragment of Bcl-6, or a shorter nt of a rabbit gue thereof, which is capable of increasing the replicative life span of a rabbit B cell.

A functional derivative of Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or of a rabbit homologue thereof, is defined as a Bcl-6, , Mcl-1, Bcl-2, A1, Bclw or Bcl2L10 protein, respectively, or a rabbit homologue thereof, which has been altered but has maintained its capability (in kind, not necessarily in amount) of increasing the replicative life span of a rabbit B cell. A functional derivative is provided in many ways, for instance through conservative amino acid substitution wherein one amino acid is tuted by another amino acid with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is not seriously affected. Alternatively, a functional derivative for instance comprises a fusion protein with a detectable label or with an ble compound.

Another aspect of the present disclosure solves the problem of efficiently introducing a nucleic acid le of interest into rabbit B cells. Contrary to expectations, the ors found that the commonly used ampho retroviral vector, which is suitable for infecting rodent cells such as murine cells and which was therefore expected to be also capable of ucing rabbit B cells, appeared not to transduce rabbit B cells efficiently; the transduction efficiency of an ampho vector at 4 days after transduction appeared lower than 1% in rabbit B cells. Therefore, the t inventors had to search for other gene delivery vehicles. Surprisingly, the inventors discovered that a gene ry vehicle which comprises the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein is capable of transducing rabbit B cells with a high efficiency, typically of 80-90% at 3-5 days after transduction. This property was quite unexpected, since a gibbon ape leukemia virus does not naturally infect rabbits. Rabbit cells were therefore not expected to contain a receptor for a GALV envelope n; the current finding was mere coincidence. Of note, transduction efficiency of human B cells with a vector containing the extracellular domain of a GALV envelope protein is typically 60-70% at 4 days after transduction, which is often lower than the transduction efficiency of rabbit B cells with this vector, despite the fact that human B cells are primate cells. This is surprising because, since an ape is also a primate, a vector containing a ased envelope protein was ed to be better capable of infecting primate cells as compared to rabbit cells. Of note, murine B cells are indeed not efficiently transduced using a vector that ses the extracellular domain of a GALV envelope protein, tent with the fact that a gibbon ape leukemia virus does not infect mice.

Now that the insight of the present disclosure has been provided that it is possible to efficiently uce rabbit B cells using at least a functional part of the extracellular domain of a GALV envelope protein, and that the transduction efficiency is even higher than the transduction efficiency of human B cells, new applications have become available. It has now become possible to introduce a nucleic acid molecule of interest into rabbit B cells with high efficiency, which is particularly ageous for producing an ex vivo rabbit B cell culture described herein. A preferred embodiment described herein is therefore a method for increasing the replicative life span of a rabbit B cell, the method comprising: - inducing, enhancing and/or maintaining expression of Bcl-6, or of a rabbit homologue thereof, in a rabbit B-cell and - inducing, enhancing and/or maintaining expression of at least one anti-apoptotic nucleic acid in said B-cell, characterized in that said rabbit B cell is provided with a c acid le encoding Bcl-6, or ng a rabbit homologue thereof, or encoding a functional part or a functional derivative thereof, and/or with at least one anti-apoptotic nucleic acid molecule, via transduction with a gene delivery vehicle that comprises the extracellular domain of a gibbon ape ia virus (GALV) envelope protein, or at least a functional part of said extracellular domain, or via transduction with a gene delivery vehicle that comprises a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, or via transduction with a gene delivery e that comprises a protein that has at least 70% sequence identity with at least a functional part of the ellular domain of a GALV envelope protein. In one preferred embodiment, said extracellular domain is of an envelope protein of GALV strain SEATO. Said extracellular domain preferably comprises the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT LTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL. Preferably, said sequence identity is at least 75%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more ably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 86%, more ably at least 87%, more preferably at least 88%, more ably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100%.

As will be understood by the skilled person, said extracellular domain, which is located at the surface (envelope) of a wild type gibbon ape leukemia virus so that it can bind a host cell, is preferably also d at the surface (envelope) of a gene delivery vehicle for transducing rabbit B cells. In one particularly preferred embodiment, a vector or other gene delivery vehicle is used that comprises an envelope protein which contains the ellular domain and transmembrane domain of a GALV envelope protein, or a onal part f, which is fused to the cytoplasmic domain of an ampho envelope protein. This allows particular efficient transduction of rabbit B cells, as shown in the Examples.

As used herein, the term "a functional part of the extracellular domain of a GALV envelope protein" means a part of said extracellular domain which is still capable of binding rabbit B cells, thereby mediating infection and/or transduction of the rabbit B cells. Such functional part may lack one, or multiple, amino acid residues which are not essential for binding, infection and/or uction of rabbit B cells.

Of course, now that the insight of the present sure has been provided, rabbit B cells can be transduced with any nucleic acid molecule of interest using at least a functional part of the extracellular domain of a GALV envelope n.

Further described is therefore an isolated or recombinant rabbit B cell bound to the extracellular domain of a GALV envelope protein, or bound to at least a functional part of said extracellular domain, or bound to a protein that has at least 70% sequence identity with at least a functional part of the ellular domain of a GALV pe protein. An isolated or recombinant rabbit B cell that is bound via at least a functional part of the extracellular domain of a GALV envelope n, or via a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein, to a gene delivery vehicle is also described herewith. In one preferred embodiment, said extracellular domain is of an envelope protein of GALV strain SEATO. Said extracellular domain preferably comprises the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT LTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL. Again, said ce identity is preferably at least 75%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more ably at least 89%, more preferably at least 90%, more ably at least 91%, more ably at least 92%, more preferably at least 93%, more preferably at least 94%, more ably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100%. Said gene delivery vehicle preferably comprises a nucleic acid molecule of interest, preferably a nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or functional derivative thereof, and/or an anti-apoptotic nucleic acid ce. With such gene delivery vehicle, a stabile rabbit B cell culture described herein can be produced. Said anti-apoptotic nucleic acid sequence is preferably an anti-apoptotic gene of the Bcl2 family, most preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, 0, and rabbit gues thereof, and functional parts and functional derivatives thereof.

A use of the extracellular domain of a GALV pe protein, or at least a functional part thereof that is capable of binding a rabbit B cell, for introducing a nucleic acid le of interest into a rabbit B cell is also herewith described, as well as a use of a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein for introducing a nucleic acid le of interest into a rabbit B cell. Further described is a use of a gene delivery vehicle comprising at least a functional part of the extracellular domain of a GALV envelope protein, or a gene delivery e comprising a protein that has at least 70% sequence ty with at least a functional part of the extracellular domain of a GALV envelope protein, said gene delivery vehicle r comprising a nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and at least one anti-apoptotic nucleic acid sequence, for increasing the replicative life span of a rabbit B cell. In one preferred embodiment, said extracellular domain is of an envelope protein of GALV strain SEATO. Said extracellular domain preferably comprises the sequence SMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT LTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL.

A preferred chimeric envelope protein, which is also used in the Examples, is shown in Figure 9. This chimeric envelope protein contains the extracellular domain of a GALV envelope protein (with the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT HGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL, indicated in bold in Figure 9) and the embrane domain of a GALV envelope protein (with the sequence LLLLLLLLILGPCII, indicated ined in Figure 9), fused to the cytoplasmic domain of an ampho envelope protein (with the sequence NRLVQFVKDRISVVQALVLTQQYHQLKPIEYEP, ted in italics and dotted-underlined in Figure 9). A vector or other gene delivery vehicle that comprises this preferred chimeric envelope protein is particularly well capable of introducing a nucleic acid molecule of interest into rabbit B cells.

Further described is therefore an isolated or recombinant rabbit B cell bound to a chimeric envelope protein as depicted in Figure 9, or to a n comprising a chimeric envelope protein as depicted in Figure 9 or to a protein that has at least 70% sequence identity with a chimeric envelope protein as depicted in Figure 9. An isolated or recombinant rabbit B cell that is bound via a ic envelope n as depicted in Figure 9, or via a protein comprising a chimeric envelope protein as depicted in Figure 9 or via a protein that has at least 70% sequence identity with a ic envelope n as depicted in Figure 9, to a vector or other gene delivery vehicle is also described herewith.

Such vector or other gene delivery e is particularly suitable for transducing rabbit B cells with a nucleic acid molecule of interest. Further described is therefore a use of a chimeric envelope protein as depicted in Figure 9, or a protein comprising a chimeric envelope protein as depicted in Figure 9 or a protein that has at least 70% ce identity with a chimeric envelope protein as depicted in Figure 9, for introducing a nucleic acid molecule of interest into a rabbit B cell.

Such vector or other gene delivery vehicle is particularly suitable for increasing the replicative life span of rabbit B cells. Further described is therefore a method for increasing the replicative life span of a rabbit B cell, the method comprising: - inducing, enhancing and/or maintaining expression of Bcl-6, or of a rabbit homologue thereof, in a rabbit B-cell and - inducing, enhancing and/or maintaining expression of at least one anti-apoptotic nucleic acid in said , characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homologue thereof, or encoding a onal part or a functional derivative thereof, and/or with at least one anti-apoptotic nucleic acid molecule, via transduction with a vector or other gene delivery vehicle that comprises a chimeric envelope protein as depicted in Figure 9, or a protein comprising a chimeric envelope protein as depicted in Figure 9, or a protein that has at least 70% sequence ty with a chimeric envelope protein as depicted in Figure 9.

Also described is a use of a gene delivery vehicle comprising a chimeric envelope protein as ed in Figure 9, or a protein comprising a chimeric envelope protein as ed in Figure 9, or a protein that has at least 70% sequence ty with a chimeric envelope protein as depicted in Figure 9, said gene delivery e further comprising a nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a functional tive thereof, and at least one anti-apoptotic c acid sequence, for increasing the replicative life span of a rabbit B cell.

It is emphasized that, although a GALV-based gene delivery vehicle is very suitable for efficient transduction of rabbit B cells with one or more nucleic acid molecule(s) of interest, such as Bcl-6 and an anti-apoptotic nucleic acid le, the use of a GALV-based gene delivery vehicle is not mandatory for obtaining rabbit B cells with a short doubling time of 20 hours or less. Other gene delivery vehicles can also be used for introducing Bcl-6 and an anti-apoptotic nucleic acid le into rabbit B cells (although the ency will often be lower), in order to produce rabbit B cells with a doubling time of 20 hours or less. As long as Bcl-6 and an anti-apoptotic nucleic acid molecule are introduced into rabbit B cells, a fastgrowing B cell culture can be obtained, although it may take longer for the lower amount of originally transduced rabbit B cells to grow out. An advantage of the use of a gene delivery vehicle that is able to efficiently transduce rabbit B cells, such as a GALV-based gene delivery vehicle as described herein, is that a higher proportion of the originally isolated B cells will be transduced, so that B cells d therefrom will be present in the resulting B cell culture. This results in a higher diversity of B cells within the B cell culture as compared to a situation wherein a gene delivery vehicle with a lower uction efficiency is used, because in the latter case a lower proportion of the original B cells are transduced. The presence of a higher diversity of B cells within the resulting B cell culture improves the chance of isolating one or more B cells with a d property. Hence, in principle, the higher the transduction ency of the gene delivery vehicle, the higher the ity of B cells within the resulting B cell culture.

In order to induce sion in rabbit B cells, a nucleic acid molecule of interest is preferably operably linked to a promoter. Non-limiting examples include a CMV promoter and a CAG promoter. In one aspect, such promoter is inducible, meaning that its ty is influenced by at least one nd, such as for instance a transcription factor.

As used herein, the term "gene delivery vehicle" means any compound capable of transferring a nucleic acid molecule into a host cell. Non-limiting examples of gene delivery vehicles e (viral) vectors and plasmids. The term "gene delivery vehicle comprising at least a functional part of the extracellular domain of a GALV envelope protein" means a gene delivery e comprising at least part of the extracellular domain of a GALV pe n, wherein said extracellular domain, or said part thereof, is capable of binding a rabbit B cell so that nucleic acid can be introduced into said rabbit B cell. As described herein , said extracellular domain, or part thereof, is preferably located at the surface of the gene delivery vehicle, so that it can bind a receptor on a rabbit B cell.

Likewise, if a gene delivery vehicle described herein comprises a protein that has at least 70% sequence identity with at least a onal part of the extracellular domain of a GALV envelope protein, said protein is ably located at the surface of the gene delivery vehicle, so that it can bind a receptor on a rabbit B cell.

The tage of identity of an amino acid or nucleic acid sequence, or the term "% sequence identity", is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art, for example "Align 2".

A GALV envelope protein is a protein that is naturally present in the viral pe of gibbon ape ia virus and that is involved in infection of host cells.

The target specificity is typically determined by the envelope protein. In one embodiment, said envelope protein is of GALV strain SEATO. Retroviral vectors containing the GALV envelope protein are known in the art and can be produced using procedures that are commonly used in the art of molecular biology, see for ce Lam et al., 1996.

The term "operably linked to a promoter" means that a nucleic acid sequence of interest is located iently close to a promoter so that the promoter can influence expression thereof. Typically, such promoter will induce or se expression of said nucleic acid of interest. The term "expression activity" refers to such induction or enhancement of expression.

As mentioned herein before, the t disclosure provides the insight that an ex vivo rabbit B cell e can be obtained with a shorter mean doubling time as compared to currently known human or murine B cell cultures. This is all the more surprising because non-rabbit compounds, such as a human Bcl-6 nucleic acid sequence, murine IL21 and human CD40L, were used in the current Examples. As shown in the Examples, the present inventors transduced rabbit B cells with a nucleic acid molecule containing a human Bcl-6 sequence and a human Bcl-xL or Mcl-1 sequence. Even though human sequences were used, and the rabbit cells were cultured in the presence of murine IL21 and human CD40L, the rabbit B cells surprisingly appeared to proliferate faster and to produce more antibody as compared to human and murine B cells.

Hence, according to the present dislosure, rabbit B cells proliferate very well using human and murine compounds. Under these reaction conditions, the rabbit B cells even proliferate better than human and murine B cells. This has amongst other things the advantage that currently used reaction conditions for human B cells do not have to be ed for rabbit B cells. There is no need to obtain rabbit IL21, rabbit CD40 or rabbit nucleic acid sequences encoding Bcl-6 or an anti- apoptotic gene. Instead, currently available human or murine compounds can be used. One embodiment described herein is a method for increasing the replicative life span of a rabbit B cell, the method sing: - inducing, enhancing and/or maintaining sion of Bcl-6, or a rabbit gue thereof, in a rabbit B-cell and - inducing, enhancing and/or maintaining sion of an anti-apoptotic nucleic acid molecule in said B-cell, characterized in that said rabbit B cell is provided with at least one nucleic acid molecule selected from the group consisting of: * a nucleic acid molecule encoding a non-rabbit Bcl-6 or a onal part or a onal derivative thereof; and * a non-rabbit anti-apoptotic nucleic acid molecule.

Preferably, said non-rabbit nucleic acid molecule is a human nucleic acid molecule because human Bcl-6 and human anti-apoptotic sequences appear to provide ularly good results in rabbit B cells. In a particularly preferred embodiment, a rabbit B cell is ed with a nucleic acid molecule encoding human Bcl-6 and with a human anti-apoptotic nucleic acid molecule, preferably human Bcl-xL or human Mcl-1 or human Bcl-2 or human A1 or human Bcl-w or human Bcl2L10.

Furthermore, a method described herein further comprising providing said rabbit B cell with IL21 and CD40L. Preferably, non-rabbit IL21 and/or bbit CD40L is used. Preferably, said IL21 is murine or human IL21, most preferably murine IL21. In r preferred ment, said CD40L is murine or human CD40L, most preferably human CD40L.

Besides increasing Bcl-6 sion and the expression of an anti-apoptotic nucleic acid molecule, it is also advantageous to induce, enhance and/or maintain expression of Blimp-1, or a rabbit homologue thereof, in a rabbit B-cell. This enhances antibody production of said B cell. Also described is a method according to the disclosure, wherein the method further comprises inducing, enhancing and/or maintaining expression of Blimp-1, or a rabbit homologue thereof, in said rabbit .

The extent of sion of Blimp-1, or of a rabbit homologue thereof, in a rabbit B cell is regulated in a variety of ways. In one embodiment a rabbit B cell is provided with a compound, which is capable of ly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue thereof. Additionally, or alternatively, a rabbit B cell is ed in the presence of a compound capable of directly or ctly increasing expression of Blimp-1, or expression of a rabbit homologue thereof. Further described is therefore a method described herein, further sing: - providing said rabbit B cell with a compound capable of directly or indirectly increasing expression of 1, or expression of a rabbit homologue thereof; and/or - culturing said rabbit B cell in the presence of a compound capable of directly or ctly increasing expression of Blimp-1, or expression of a rabbit homologue thereof.

Said compound capable of increasing expression of Blimp-1, or of a rabbit homologue thereof, most preferably comprises IL21. Hence, in one preferred embodiment described herein, rabbit B cells are cultured in the presence of IL21, at least during part of the culture time.

In another embodiment said compound capable of increasing Blimp-1 expression comprises a Signal Transducer of Activation and Transcription 3 (STAT3) protein or a functional part or a functional derivative thereof, and/or a nucleic acid molecule coding therefore. STAT3 is a signal transducer, which is involved in B cell development and differentiation. STAT3 is capable of upregulating Blimp-1 expression. In one red embodiment, a rabbit B cell is provided with a c acid molecule ng STAT3 or a functional part or a functional derivative thereof, wherein the expression of said nucleic acid molecule is regulated by an exogenous inducer of repressor, so that the extent of STAT3 expression is regulated at will. For instance, one of the earlier ned inducible expression systems is used. In one embodiment a fusion product comprising STAT3, or a functional part or a functional derivative, and ER is used. For instance, a rabbit B cell is provided with a nucleic acid molecule encoding an estrogen or (ER) and STAT3 as a fusion protein ER-STAT3. This fusion protein is inactive because it forms a complex with heat shock proteins in the cytosol. This way, STAT3 is unable to reach the nucleus and Blimp-1 expression is not enhanced. Upon administration of the exogenous inducer 4 hydroxy-tamoxifen (4HT), the fusion n ER-STAT3 iates from the heat shock proteins, so that STAT3 is capable of entering the nucleus and activating Blimp-1 expression.

As used herein, a functional part of STAT3 is defined as a fragment of STAT3 that has the same capability - in kind, not necessarily in amount - of increasing expression of Blimp-1, or of a rabbit homologue thereof, as compared to natural STAT3. Such functional part is for instance devoid of amino acids that are not, or only very little, involved in said capability.

A functional derivative of STAT3 is defined as a STAT3 protein, which has been altered but has maintained its capability (in kind, not arily in amount) of sing expression of Blimp-1, or of a rabbit homologue thereof. A onal derivative is provided in many ways, for instance through conservative amino acid substitution wherein one amino acid is substituted by another amino acid with generally similar properties (size, hobicity, etc), such that the l functioning is not sly ed. Alternatively, a functional derivative for instance comprises a fusion protein with a detectable label or with an inducible Since STAT3 is capable of increasing expression of Blimp-1, or increasing expression of a rabbit homologue thereof, it is also possible to indirectly se expression of Blimp-1, or of a rabbit homologue thereof, by administering a compound capable of increasing the activity and/or expression of STAT3. In one embodiment, a rabbit B cell is therefore provided with a compound that is capable of enhancing the activity of STAT3, so that expression of Blimp-1, or of a rabbit homologue thereof, is indirectly enhanced.

STAT3 is activated in a variety of ways. Preferably, STAT3 is activated by providing a rabbit B cell with a cytokine. nes, being lly involved in B cell differentiation, are very effective in ting STAT proteins. Very effective activators of STAT3 are IL21 and IL6, but also IL2, IL7, IL10, IL15 and IL27 are known to activate STAT3. Moreover, Toll-like ors (TLRs), which are involved in innate immunity, are also capable of activating STAT3. One embodiment relates to a method described herein, wherein said rabbit B cell is cultured in the presence of IL21, IL2, IL6, IL7, IL10, IL15 and/or IL27. Most preferably IL21 is used, since IL21 is particularly suitable for enhancing antibody production of rabbit B cell es described . IL21 is capable of upregulating Blimp-1 expression, even when Blimp-1 expression is counteracted by BCL6.

Additionally, or alternatively a mutated Janus kinase (JAK) , or a mutated rabbit homologue of a JAK, is used in order to activate STAT3. Naturally, a JAK is capable of phosphorylating STAT3 after it has itself been activated by at least one cytokine. A mutated Janus kinase, or a mutated rabbit homologue of a JAK, capable of activating STAT3 independently of the presence of cytokines, is particularly suitable in a method described .

In yet another ment, expression of 1, or of a rabbit homologue thereof, is sed by providing a rabbit B cell with a suppressor of cytokine signalling (SOCS) n, or a rabbit homologue thereof, and/or by activating a SOCS protein or a rabbit homologue thereof within said cell. Alternatively, or additionally, at least one of the E-proteins E47, E12, E2-2 and HEB is used in order to increase expression of Blimp-1, or of a rabbit homologue thereof. E47 is a transcription factor that belongs to a family of helix-loop-helix proteins, named E- proteins. There are four E-proteins, E12, E47, E2-2 and HEB, which are involved in lymphocyte development. E12 and E47 are d by one gene, named E2A, which is spliced differently. E proteins have been described as tumor suppressors.

One of the specific targets of E47 are the Socs1 and Socs3 genes.

Also described is a method according to the present disclosure, r increasing expression of Blimp-1, or of a rabbit gue thereof, in a rabbit B cell by providing said B cell with a compound capable of directly or indirectly increasing expression of Blimp-1, or of a rabbit gue thereof, and/or culturing said B cell in the presence of a compound capable of ly or indirectly increasing expression of Blimp-1, or of a rabbit homologue thereof, wherein said compound comprises: - STAT3 or a functional part or a functional derivative thereof, and/or - a compound capable of activating STAT3, and/or - a compound capable of enhancing expression of STAT3, and/or - IL21, IL2, IL6, IL7, IL10, IL15, IL27, a SOCS protein, one of the eins E47, E12, E2-2 or HEB, a mutated Janus kinase and/or a nucleic acid sequence ng STAT3 , or a rabbit homologue or a functional part or a functional derivative thereof.

Most preferably, said compound is IL21.

Also described is isolated or recombinant rabbit B cells obtainable with a method described herein. Such isolated or inant rabbit B cells preferably comprise an exogenous anti-apoptotic nucleic acid sequence and an exogenous nucleic acid sequence encoding Bcl-6 , or a rabbit homologue f, or a onal part or a functional tive thereof. Further described is therefore an isolated or recombinant rabbit B cell, comprising an exogenous nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a onal part or a functional derivative thereof, and an exogenous anti-apoptotic nucleic acid sequence. As explained before, said exogenous nucleic acid molecule either contains a nucleic acid sequence that does not naturally occur in rabbit B cells, or an additional copy of a natural rabbit B cell nucleic acid sequence. Bcl-xL, Mcl-1, Bcl- 2, A1, Bcl-w and Bcl2L10, and rabbit homologues thereof, are preferred antiapoptotic nucleic acid molecules. One preferred aspect therefore describes an ed or recombinant rabbit B cell, which comprises an exogenous nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a onal derivative thereof, and an exogenous nucleic acid sequence encoding BclxL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a rabbit homologue thereof, or a functional part or a functional derivative thereof.

Said nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a functional derivative f, and said exogenous antiapoptotic nucleic acid sequence may be t on one nucleic acid molecule. atively, these sequences are present on at least two different nucleic acid molecules.

Preferably, non-rabbit sequences are used, as explained before. A preferred embodiment therefore describes an isolated or recombinant rabbit B cell comprising a non-rabbit anti-apoptotic nucleic acid sequence and a non-rabbit nucleic acid sequence encoding Bcl-6, or a rabbit homologue thereof, or a functional part or a onal derivative thereof. Said non-rabbit nucleic acid sequence preferably contain human sequences.

In a particularly preferred embodiment, an isolated or recombinant rabbit B cell is provided which comprises: - a c acid sequence encoding human Bcl-6 or a functional part or a functional derivative thereof, and - a human poptotic nucleic acid sequence, ably ng human Bcl-xL or human Mcl-1 or human Bcl-2 or human A1 or human Bcl-w or human Bcl2L10, or a functional part or a onal derivative thereof. Again, said nucleic acid sequence encoding Bcl-6 or a functional part or a functional derivative thereof, and said anti-apoptotic nucleic acid sequence, may be present on one nucleic acid molecule, or, alternatively, these sequences may be present on at least two different nucleic acid molecules.

Also described are ex vivo rabbit B cell cultures obtainable by the methods described herein. An important advantage is the fact that ex vivo B cell cultures are now ed with a short mean doubling time. Described is therefore an ex vivo rabbit B cell culture which has a mean doubling time of 20 hours or less. A further preferred embodiment describes an ex vivo rabbit B cell culture sing rabbit B cells described herein. Said rabbit B cells ably comprise a nucleic acid sequence ng human Bcl-6 or a functional part or a functional derivative thereof, and an anti-apoptotic nucleic acid sequence. Also described is an ex vivo rabbit B cell culture comprising rabbit B cells in the presence of non-rabbit IL21 and/or bbit CD40L. Preferably, said IL21 is murine or human IL21, most preferably murine IL21. In another preferred embodiment, said CD40L is murine or human CD40L, most preferably human CD40L.

An antibody when obtained by a method described herein is also described herewith, as well as an antibody produced by a rabbit B cell described herein or by an ex vivo rabbit B cell culture bed herein. Such antibody is particularly useful for therapeutic or diagnostic applications. Preferably, said antibody is a onal antibody.

The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

In this ication where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated ise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the ption in this specification reference may be made to subject matter that is not within the scope of the claims of the t application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Examples Example 1 Transduction of B cells Gene transfer into lymphocytes by traditional methods like calcium phosphate precipitation, liposome formation or electroporation is inefficient but more importantly stable gene integration is generally absent. Viral transduction r leads directly to stable gene integration into the genome of the target cell and can be very efficient if the proper virus envelope is chosen. Both retroviral and lentiviral transductions are suitable for efficient gene er. While retroviral integration is dependent on cell division, lentiviral transduction can also be applied to non-dividing cells like plasma B cells. scale ation of recombinant retrovirus can easily be achieved by using stable producer cell lines such as the Phoenix expression rm (Kinsella and Nolan, 1996). Production of high titer lentivirus tends to be more cumbersome mainly because of the toxicity of the expressed virus proteins and envelopes.

For the current Examples, we used a Moloney Murine Leukemia Virus (MMLV) based rm, using either amphotropic or Gibbon Ape Leukemia Virus (GALV) envelope expressing producer cells (Wilson et al., 1995). In our GALV-based vector, the transmembrane domain of the GALV strain SEATO envelope protein was fused to the cytoplasmic domain of an ampho envelope protein (Figure 9).

The transfer vector is set-up such that Bcl-6, Bcl-xL and the green fluorescent protein (GFP) marker protein are simultaneously translated from the same viral RNA (Figure 8). This multicistronic approach is achieved by placing a ‘self-cleaving’ 2A peptide sequence (Szymczak et al., 2004) between the BCL-6 and BCL-xL coding regions and an al mal Entry ce (IRES) am of the GFP reporter gene. Viral transduction efficiencies are high and unbiased.

Generation of immortalized rabbit B cells Human memory B cells were immortalized using the BCL-6 / Bcl-xL technology described by Kwakkenbos et al., 2010 and patent ation . In brief, PBMC’s from rabbit blood were isolated using a ficoll density gradient and stained for Ig expression using an antibody that recognizes Ig (IgG H+L: IgG heavy chain and kappa and lambda light chains) mes in combination with an IgM specific antibody. B cells were isolated (Ig positive, or Ig positive + IgM negative) using a FACS sorter and stimulated on γ irradiated (50 Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L- L cells, 105 cells ml−1) er with recombinant mouse interleukin (IL)-21 for 36-48 hours. Cells were harvested and washed with medium without FCS and cells were then transferred to Retronectin® (Takara, Shiga, Japan)- coated tissue culture plates where they were transduced with a retroviral vector containing BCL-6, Bcl-xL, and GFP as a reporter protein.

Alternatively cells were transduced with a retroviral vector containing BCL-6, Mcl- 1 and GFP. Transduced B cells were maintained in culture with CD40 Ligand expressing L-cells and IL-21. In Figure 1 the transduction efficiency is compared for GALV and amphotropic type retroviruses at 4 days after transduction. Four days after transduction with the amphotropic type retrovirus 0.8% of the cells was transduced compared to 80% of cells after uction with a GALV type retrovirus. Clearly the GALV type retrovirus is superior to the amphotropic type retrovirus for transducing rabbit B cells.

Example 2 Cell culture.

We maintained B cells (2 × 105 cells ml−1) in Iscove’s ed Dulbecco’s medium (Gibco) containing 8% FBS and penicillin- streptomycin (Roche) supplemented with recombinant mouse interleukin 21 (IL-21) (50 ng ml−1) and cultured them on γ irradiated (50 Gy) mouse L cell fibroblasts stably expressing CD40L - L cells, 105 cells ml−1). To determine cell doubling time cells were cultured in l plates at 50-100.000 cells/well together with CD40L- L cells and IL-21. Every 3-4 days cell were counted and 50-100.000 cells transferred to a new well. In Figure 2 growth curves are depicted for B cells from two human donors (89 and 93), one llama B-cell sample (Llama) and one rabbit B-cell sample which was transduced with a GALV type retrovirus carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Bcl-xL (Rb 6XL). Also a growth curve is depicted for one rabbit sample that was transduced with a GALV type irus carrying a nucleic acid molecule containing a human Bcl-6 ce and a human Mcl-1 (Rb 6M). The transduced rabbit B cells have an average doubling time of 19 hours and thus grow faster than the human or llama B cells that have doubling times between 26 and 32 hours. These average doubling times were originally calculated by determining the increase of B cells during several 3-4 days time intervals, and averaging the obtained results. Subsequently, the overall average doubling time during the whole culturing period was calculated. This resulted in an average doubling time of the transduced rabbit B cells of 18 hours, an average doubling time of the transduced human B cells of 25-29 hours and an average doubling time of the transduced llama B cells of 27 hours. This ms our ations that our methods yield rabbit B-cell cultures with a mean doubling time of 20 hours or less, whereas human, murine and llama B cells typically have a doubling time of between 25 and 36 hours.

Example 3 B-cell receptor expression and antigen-specific staining Immortalized human B cells express the B-cell receptor. This quality s antigen-specific staining and sorting of B cells. To determine whether the B-cell receptor is also expressed on transduced rabbit B cells, B-cell clones are d with scently labeled antibodies reacting specifically with either rabbit IgG, rabbit IgM or rabbit IgA. B cells were washed in cold (4°C) cell culture medium and incubated on ice in the dark with cell culture medium containing immunofluorescently ed antibodies that are specific for either rabbit IgG, IgM, IgA or ed antigen. Afterwards excess of labelled antibodies or antigen was washed away and B-cell receptor expression analysed on a FACS analyser; the Guava easycyte (Millipore) or FACS Aria3 (BD).

In Figure 3 three different B-cell clones of different isotypes were stained with fluorescently ed antibodies specifically recognizing rabbit antibody isotype IgG, IgA or IgM. Clearly the B-cell receptor can be efficiently stained for the different rabbit antibody isotypes. We therefore conclude that immortalized rabbit B cells also express the B-cell or.

In addition, also fluorescently labeled nza proteins were used to stain for nza-specific B-cells from s that had been zed with a human influenza vaccine or untreated control rabbits e 4). Rabbit B cells were stained with fluorescently d H1, H3 or influenza B and sorted 1 cell per well using a FACS sorter.

Example 4 Development of single-cell derived, clonal rabbit B cell cultures.

Transduced B cells were sorted one cell per well using a FACS sorter and cultured in the presence of γ irradiated (50 Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L- L cells, 105 cells ml−1) together with recombinant mouse IL-21.

Every 3-4 days fresh CD40L- L cells and IL-21 were added. Starting 9 days after seeding the cells (one cell per well), the supernatants were analyzed in ELISA for the production of rabbit immunoglobulin G (IgG). For comparison also the human IgG in the atant of human B-cell clones was analyzed in parallel.

In Figure 7 the antibody concentration in the supernatant is depicted over time starting at 9 days after the initiation of the single cell cultures. The antibody concentration was determined for two human donors and one rabbit B-cell sample that were transduced with a GALV type retrovirus carrying a nucleic acid le containing a human Bcl-6 sequence and a human Bcl-xL and for one rabbit B-cell sample that was transduced with a GALV type retrovirus carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Mcl-1. B cell clones from rabbits produce IgG concentrations of 30 ng/ml and 100 ng/ml within a shorter time period (9-10 days and 11-12 days, respectively) than do the human B- cell clones (13-18 and 15-20 days, respectively). This provides the important advantage that it allows for earlier screening for antibodies of interest of rabbit B cell , compared to human B cell clones.

Example 5 Immunization of rabbits. 2 New d White rabbits were immunized with a human nza vaccine containing 15ug H1N1, 15ug H3N2 and 15ug infl B in complete Freunds adjuvans.

After 3 weeks rabbits were boosted with the same vaccine in incomplete Freunds adjuvans. Five days after the boost rabbits were bled, B-cells were isolated from the blood and transduced with a GALV type irus (containing the extracellular domain and transmembrane domain of the GALV strain SEATO envelope protein, fused to the cytoplasmic domain of an ampho envelope protein) carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Bcl-xL. Transduced B cells were seeded at different cell densities into culture plates and cultured as described in Example 4. Also, transduced B cells were labeled with fluorescently labeled components of the vaccine; H1, H3 or influenza B and sorted 1 cell per well using a FACS sorter and cultured as described in Example 4. The supernatants of the cultured cells were analyzed for binding to the complete vaccine or to its dual components. The results are depicted in Figures 4-6 and show that antigen-specific B cells can be identified in the B-cell pool from vaccinated s by seeding cells at ent y (Figure 5) and also very efficiently by sorting cells using the labeled antigens (Figure 4 and Figure Example 6 Rabbit B cells are alized by the introduction of the genes Bcl-6 and Bcl-xl using an ropic type retrovirus.

Immortalization of rabbit B cells by introduction of the genes Bcl-6 and Bcl-xl can be achieved by using different types of vectors, such as for instance GALV and amphotrophic type retroviruses as is shown in Example 1. The growth of B cells transduced with the amphotrophic type retrovirus was further pursued to confirm that introduction of Bcl-6 and Bcl-xl by amphotrophic retrovirus also leads to immortalization of rabbit B cells. Four days after uction with the amphotropic type retrovirus 0.8% of the cells was transduced compared to 80% of cells after transduction with a GALV type retrovirus (Figure 1 and Figure 10). Ten days after transduction 94% of the cell population transduced with the amphotrophic retrovirus was GFP positive demonstrating that the transduced cells ow the non-transduced cells (Figure 10).

To determine cell doubling time cells were cultured as done in Example 2 in 24- well plates at 50-100.000 cells/well together with CD40L- L cells and IL-21. Every 3-4 days cell were counted and 50-100.000 cells transferred to a new well. In Figure 11 the growth curve is depicted for rabbit B cells transduced with amphotrophic virus. The calculated doubling time is 19 hours, which is able to rabbit B cells transduced with GALV type retrovirus (18 hours). In conclusion, introduction of Bcl-6 and Bcl-xl into rabbit B cells by rophic retrovirus also results in alization of rabbit B cells, although the transduction efficiency is much lower as compared to a GALV based vector.

Brief description of the gs Figure 1.

Transduction of rabbit memory B cells. Rabbit B cells were isolated from PBMCs based on Ig expression. Cells were activated for 36-40hrs on CD40L L-cells with rm-IL-21. Cells were transduced with a retroviral vector containing BCL6 and Bcl- xL. Both GALV and amphotropic type retroviruses were . Transduced cells are then cultured on CD40L-L cells in the presence of recombinant mouse IL-21.

After four days of e the transduction efficiency was determined based on GFP expression. GALV typed retrovirus showed superior (80%) transduction efficiency compared to amphotropic (0.8%) typed irus.

Figure 2.

Growth curves were analyzed for rabbit B cells transduced with a retroviral vector containing BCL6 and Bcl-xL or a retroviral vector containing BCL6 and Mcl-1. For comparison growth curves were analysed in parallel B cells from llama cells and human cells from two different donors that were transduced with an identical retroviral vector containing BCL6 and Bcl-xL. Figure 2 also shows transduced rabbit B cells grow y.

Doubling time: Human B cells: 25-29 hrs Rabbit B cells 6XL: 18hrs Rabbit B cells 6M: 18hrs Llama: 27 hrs.

Figure 3.

IgG, IgM and IgA surface immunoglobulin expression was detected using FACS on three different Bcl-6 Bcl-xL transduced rabbit B-cell clones.

Figure 4.

Identification of antigen-specific rabbit B-cells within a pool of rabbit B cells with ent specificities.

Figure 5.

Antigen-specific rabbit antibodies were obtained against the different components of a human influenza vaccine containing 15ug H1N1, 15ug H3N2 and 15ug infl B .

Rabbits were immunized and boosted with the human influenza vaccine. B cells were immortalized and seeded at different ies in 384-well plates on CD40L-L cells in the presence of inant mouse IL-21. Antibodies present in the rabbit B cell e supernatants were screened in ELISA for influenza-specificity.

Antigen-specific antibodies were observed for all the components of the vaccine.

Figure 6.

Immortalized B cells from rabbits immunized with a human influenza vaccine containing 15ug H1N1, 15ug H3N2 and 15ug infl B were stained with fluorescently labelled nza proteins. B cells showing binding to the influenza proteins were sorted 1 cell per well in 384-well plates on CD40L-L cells in the presence of inant mouse IL-21 using a FACSAria sorter. Supernatants were screened in ELISA for nza-specific antibodies. Antigen-specific antibodies were ed with a high frequency for the components of the e that were used for antigenspecific sorting.

Figure 7.

Antibody concentration in the supernatant of clonal B cells at different time points.

Human, llama and rabbit transduced B cells were seeded 1 cell per well in the presence of ated CD40L- L cells and supplemented with mouse IL-21. Every 3-4 days CD40L- L cells and IL-21 were replenished. The IgG concentration was analyzed in ELISA for individual wells at different time points during culture.

Each measurement was done on different wells. The rabbit B cells were either transduced with a retroviral vector containing BCL6 and Bcl-xL or a retroviral vector containing BCL6 and Mcl-1. All other cells (human and llama) were transduced with BCL6 and Bcl-xL.

Figure 8.

Schematic representation of the vector used to transduce the rabbit and human B cells Figure 9.

Sequence of the extracellular domain of GALV SEATO envelope protein (bold) and the transmembrane domain of the GALV SEATO envelope protein (underlined), fused to the cytoplasmic domain of ampho envelope protein (italics + dottedunderlined Figure 10.

Transduction of rabbit memory B cells and outgrowth of rabbit B cells transduced with amphotrophic type retrovirus. Rabbit B cells were isolated from PBMCs based on Ig expression. Cells were activated for 36-40hrs on CD40L L-cells with rm-IL-21. Cells were uced with a retroviral vector containing BCL6 and Bcl- xL. Both GALV and amphotropic type retroviruses were tested. Transduced cells were then cultured on CD40L-L cells in the presence of recombinant mouse IL-21.

After four days of culture the transduction efficiency was determined based on GFP expression. GALV typed retrovirus showed superior (80%) transduction efficiency compared to ropic (0.8%) typed retrovirus. After 10 days 94% of rabbit B cells transduced with amphotrophic type irus were immortalized based on GFP expression showing wth of transduced cells over non-transduced cells.

Figure 11.

A growth curve was analyzed for rabbit B cells transduced with a amphotrophic type iral vector containing BCL6 and Bcl-xL. Figure 11 also shows Rabbit cells transduced with ampho-coated virus grow similarly fast as cells transduced by oated virus.

Doubling time: Rabbit B cells Ampho 6XL: 19 hrs.

References Christopherson, K.S. et al. PNAS 89, 6314-8 (1992) Guzman, L. M. et al. Bacteriol 177, 4121–4130 (1995) T.M. Kinsella, G.P. Nolan, Hum Gene Ther 7 (1996) 1405.

Kwakkenbos et al. Generation of stable monoclonal antibody-producing B cell receptor- positive human memory B cells by genetic programming. Nature Medicine (2010) vol. 16 (1) pp. 123-8 Lam et al. Improved gene transfer into human lymphocytes using retroviruses with the gibbon ape leukemia virus envelope. Human gene therapy 7 (1996) 1415-1422 A.L. Szymczak, C.J. Workman, Y. Wang, K.M. i, S. lou, E.F. Vanin, D.A.A. Vignali, Nat hnol 22 (2004) 589.

C.A. Wilson, M.V. Eiden, W.B. Anderson, C. Lehel, Z. Olah, J Virol 69 (1995) 534.

Claims

1. A method for ing an ex vivo B cell culture with a mean doubling time of 20 hours or less, the method sing: 5 - inducing, enhancing or maintaining sion of Bcl-6 in a B cell, - inducing, enhancing or maintaining sion of at least one antiapoptotic nucleic acid molecule comprising a gene of the Bcl-2 family in said B cell, 10 characterized in that said B cell is a rabbit B cell.

2. A method for increasing the replicative life span of a rabbit B cell, the method comprising: - ng, enhancing or maintaining expression of Bcl-6 in a rabbit B 15 cell, and - inducing, enhancing or maintaining expression of at least one antiapoptotic nucleic acid comprising a gene of the Bcl-2 family in said B cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, with at least one anti-apoptotic nucleic acid 20 molecule sing a gene of the Bcl-2 family, or with a combination thereof, via transduction with a gene delivery vehicle that comprises the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein.

3. Use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, for introducing a c acid molecule encoding Bcl-6 and at least one anti- tic c acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell.

4. A method for obtaining antibodies, comprising: 5 - inducing, enhancing or maintaining expression of Bcl-6 in a rabbit B cell; - ng, ing or maintaining expression of at least one antiapoptotic nucleic acid molecule comprising a gene of the Bcl2- family in said B cell; 10 - culturing said B cell ex vivo; and - harvesting antibodies produced by said B cell within 7-14 days.

5. The method of claim 4, wherein antibodies produced by said B cell are harvested within 9-12 days.

6. The method according to any one of claims 1-2, and 4-5, characterized in that said rabbit B cell is provided with: * a nucleic acid le ng a non-rabbit Bcl-6, or * at least one non-rabbit anti-apoptotic nucleic acid molecule 20 comprising a gene of the Bcl-2 family, or a combination thereof.

7. The method according to claim 6, wherein said non-rabbit nucleic acid molecule is an anti-apoptotic nucleic acid molecule comprising a human 25 gene of the Bcl-2 family or a nucleic acid molecule encoding a human Bcl-6.

8. The method according to any one of claims 1-7, wherein said gene of the Bcl-2 family is selected from the group consisting of Bcl-xL, Mcl 1, Bcl-2, 30 A1, Bcl-w and Bcl2L10.

9. The method according to any one of claims 1-2 and 4-8, wherein the method also comprises inducing, enhancing or maintaining expression of Blimp 1 in said rabbit B cell.

10. The method according to any one of claims 1-2 and 4-9, further comprising providing said rabbit B cell with IL21 and CD40L.

11. The method according to claim 10, n said IL21 is mouse or 10 human IL21, or wherein said CD40L is mouse or human CD40L, or a ation thereof.

12. The method according to any one of claims 1-2 and 4-11, further comprising: 15 - providing said rabbit B cell with a nucleic acid molecule encoding STAT3; or- culturing said rabbit B cell in the presence of IL21, or a combination thereof.

13. A rabbit B cell, which is bound via the extracellular domain of a 20 GALV envelope protein, or via a protein that has at least 70% sequence ty with the extracellular domain of a GALV envelope protein, to a gene ry vehicle that comprises a nucleic acid sequence encoding Bcl-6 and an anti-apoptotic c acid sequence comprising a gene of the Bcl-2 family.

14. The rabbit B cell according to claim 13, wherein said anti-apoptotic nucleic acid sequence is a nucleic acid sequence encoding a protein selected from the group consisting of Bcl xL, Mcl-1, Bcl-2, A1, Bcl w, Bcl2L10, and any combination thereof.

15. An isolated or recombinant rabbit B cell comprising: - a non-rabbit anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family, and - a bbit nucleic acid molecule encoding Bcl-6.

16.he isolated or recombinant rabbit B cell according to claim 15, wherein the gene of the Bcl-2 family s Bcl-xL, Mcl-1, Bcl-2, A1, Bcl w, or Bcl2L10. 10

17. The rabbit B cell according to claim 15 or 16, wherein said non-rabbit nucleic acid le comprises a human gene of the Bcl-2 family, or encodes human Bcl-6, or a combination thereof.

18. An ex vivo rabbit B cell culture comprising the rabbit B cells 15 ing to any one of claims 13-17, which has a mean doubling time of 20 hours or less.

19. An ex vivo rabbit B cell culture when obtained by a method according to any one of claims 1 and 6-12.

20. Use of a gene delivery vehicle comprising the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV pe protein, and a nucleic acid sequence encoding Bcl-6 and at least 25 one anti-apoptotic nucleic acid sequence comprising a gene of the Bcl-2 family, for increasing the replicative life span of a rabbit B cell.

21. The method according to claim 2, the use according to claim 3, or the rabbit B cell according to claim 13 or 14, or a use according to claim 20, wherein said extracellular domain is of an envelope protein of GALV strain SEATO.

22. The method according to claim 2, or the rabbit B cell according to 5 claim 13 or 14, or the use according to claim 20, wherein said gene delivery vehicle comprises a chimeric envelope protein as depicted in